Study on the Sleep-Improvement Effects of Hemerocallis Citrina

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Article Study on the Sleep-Improvement Effects of Hemerocallis citrina Baroni in Drosophila melanogaster and Targeted Screening to Identify Its Active Components and Mechanism

Yuxuan Liang, Riming Huang, Yongchun Chen, Jing Zhong, Jie Deng, Ziyi Wang, Zhuojun Wu, Meiying Li, Hong Wang and Yuanming Sun *

College of Food Science, South China Agricultural University, Guangzhou 510642, China; [email protected] (Y.L.); [email protected] (R.H.); [email protected] (Y.C.); [email protected] (J.Z.); [email protected] (J.D.); [email protected] (Z.W.); [email protected] (Z.W.); [email protected] (M.L.); [email protected] (H.W.) * Correspondence: [email protected]

Abstract: Hemerocallis citrina Baroni (HC) is an edible plant in Asia, and it has been traditionally used for sleep-improvement. However, the bioactive components and mechanism of HC in sleep- improvement are still unclear. In this study, the sleep-improvement effect of HC hydroalcoholic extract was investigated based on a caffeine-induced insomnia model in Drosophila melanogaster (D. melanogaster), and the ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-ESI-Orbitrap-MS) and network pharmacology strategy were further combined to screen systematically the active con- Citation: Liang, Y.; Huang, R.; Chen, stituents and mechanism of HC in sleep-improvement. The results suggested HC effectively regu- Y.; Zhong, J.; Deng, J.; Wang, Z.; Wu, lated the number of nighttime activities and total sleep time of D. melanogaster in a dose-dependent Z.; Li, M.; Wang, H.; Sun, Y. Study on manner and positively regulated the sleep bouts and sleep duration of D. melanogaster. The target the Sleep-Improvement Effects of screening suggested that quercetin, luteolin, kaempferol, caffeic acid, and nicotinic acid were the Hemerocallis citrina Baroni in main bioactive components of HC in sleep-improvements. Moreover, the core targets (Akt1, Cat, Drosophila melanogaster and Targeted Ple, and Sod) affected by HC were veriﬁed by the expression of the mRNA of D. melanogaster. In Screening to Identify Its Active summary, this study showed that HC could effectively regulate the sleep of D. melanogaster and Components and Mechanism. Foods further clariﬁes the multi-component and multi-target features of HC in sleep-improvement, which 2021, 10, 883. https://doi.org/ 10.3390/foods10040883 provides a new insight for the research and utilization of HC.

Academic Editor: Massimo Castellari Keywords: Hemerocallis citrina Baroni; liquid chromatography coupled with mass spectrometry sleep; Drosophila melanogaster; network pharmacology; mRNA expression Received: 12 March 2021 Accepted: 15 April 2021 Published: 17 April 2021 1. Introduction Publisher’s Note: MDPI stays neutral Sleep is a complex physiological process influenced by many factors [1]. The sleep with regard to jurisdictional claims in disorders of the population keep increasing in modern society because of the changes in published maps and institutional affil- lifestyle and environment [2]. Emerging evidence indicates that inadequate sleep can have iations. many negative effects on health, such as obesity, heart disease, anxiety, and depression [3–7]. Epidemiological studies have shown that the prevalence of sleep disorders ranges from 3.9% to 22.0% in the world population [8–11]. Drug therapy is still the main method of sleep- improvement; however, more and more studies have paid attention to dietary supplements Copyright: © 2021 by the authors. in avoiding dependence, addiction, and other side-effects induced by drugs [2,12–15]. Licensee MDPI, Basel, Switzerland. Similar to mammalian sleep, circadian rhythms of D. melanogaster have been quantified This article is an open access article as a sleep/wake activity and neuromodulation of sleep behavior [16], and the locomotor distributed under the terms and activity of D. melanogaster is often used to measure sleep/wake activity [17]. At present, conditions of the Creative Commons the spontaneous locomotor activity of D. melanogaster is quantified using a PC-based Attribution (CC BY) license (https:// locomotor activity monitoring system, which records the locomotor activity of individual creativecommons.org/licenses/by/ D. melanogaster based on the interruption of an infrared beam. It is able to monitor the 4.0/).

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locomotor behavior of D. melanogaster over a longer period of time under standardized living conditions, as well as to measure the sleep parameters of D. melanogaster [18]. The previous studies have defined the duration of sleep, the number of sleep cycles, and the average duration of each sleep in Drosophila [13,19,20]. Therefore, models of D. melanogaster have been valuable in the discovery of novel sleep regulators [17]. Hemerocallis citrina Baroni (HC), as an edible plant, is widely grown in Asia with high economic value due to its good taste and health-promoting properties, and it has a long history of being used to ameliorate sleep-related disorders [21–23]. However, the unclear material basis and molecular mechanisms of HC in sleep-improvement re- strict its further development and application. Phytochemical analysis revealed that HC contains many bioactive components such as flavonols, anthraquinones, and polyphenols [24]. Furthermore, previous studies have shown that HC possesses anti-depression and anti-inflammatory effects [23,25–27]. Among them, Du et al. reported that the main compounds responsible for its antidepressant activity are flavonoids (especially rutin and hesperidin) [22,28]. Furthermore, a few studies have indicated that some single compounds such as rutin, hyperoside, and caffeic acid might possess the potential function of improving sleep [29–33], though these studies mainly focused on the bioactivity of a single compound. However, food provides a characteristic combination of multiple nutrients in one group that work together to play an essential role. Therefore, we speculate that there are multiple compounds in HC that play a role in sleep-improvement. With the development of pharmacology, bioinformatics, and network science, Hopkins proposed the concept of “network pharmacology” in 2008 [34], which provides novel insights to reveal the coordinated interaction among multi-components, multi-targets, and multi-pathways, and to deepen the understanding of complex diseases at the system level [35,36]. So, in this study, the sleep-improvement effect of HC was firstly investigated based on the caffeine-induced insomnia model in D. melanogaster. Then, high-resolution UHPLC-ESI-Orbitrap-MS was used to analyze the composition of HC. On this basis, the network pharmacology strategy was used to screen bioactive compounds and identify the mechanism of HC in sleep-improvement. Moreover, the transcription levels of core targets of the network were further verified.

2. Materials and Methods 2.1. Materials Commercial Hemerocallis citrina Baroni (ﬂower buds) was obtained from Qidong county, Hunan province. Acetonitrile hypergrade for liquid chromatography coupled with mass spectrometry (LC-MS) LiChrosolv® was bought from Merck KgaA (Darmstadt, Germany), and methanol hypergrade was purchased from SIMARK (Tianjin, China). Ultra- pure water was prepared using the Unique-R10 system from Research Water Puriﬁcation Technology Co. Ltd. (Xiamen, China).

2.2. Preparation of HC Extract Two hundred grams of HC was ground to powder and extracted three times in 2000 mL of 60% ethanol under a temperature of 25 ◦C, each time for 24 h. The combined ethanolic extract was centrifuged at 12,000 rpm for 15 min, and then the supernatant was concentrated under reduced pressure on a rotary evaporator (45 ◦C, 60 hpa). Part of the concentrate was lyophilized for 72 h to obtain lyophilized powder and stored at −20 ◦C. Additionally, part of the concentrate was ﬁltered by a 0.22 µm ﬁlter membrane and then stored at −4 ◦C until UPLC-orbitrap-MS analysis.

2.3. Drosophila Stocks Wild-type D. melanogaster (Canton-S strain) were purchased from the Core Facility of Drosophila Resources and Technology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The ﬂies were raised in standard medium (5% sucrose, 8% Foods 2021, 10, 883 3 of 15

cornmeal, 4% dried yeast, 1% agar, and 0.5% propionic acid) at 25 ◦C in 12:12 h light: dark cycles. Male ﬂies (3-day-old) were collected under CO2 anesthesia for formal experiment.

2.4. Measurement of Locomotor Activity The food used in the measurement of locomotor activity was made up of 1% agar and 5% sucrose. Agar-sucrose food containing 0.1% caffeine was made. On this basis, 0.625%, 1.250%, and 2.500% of HC extract were add to the agar-sucrose food containing 0.1% caffeine, respectively. Each group contained 19 fruit ﬂies. D. melanogaster were individually transferred in separate plastic tubes and were monitored through the Drosophila Activity Monitoring System (DAM; TriKinetics, Waltham, MA, USA). Monitoring was performed for 3 days under conditions of constant darkness at 25 ◦C and 60% relative humidity, and locomotor activity was taken every minute. The sleep of D. melanogaster was deﬁned as no locomotor activity observed within 5 min [37], and the number of sleep bouts and sleep duration was counted by excel [13,38].

2.5. UHPLC-Orbitrap-MS Analysis A Q Exactive-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Ger- many), equipped with an electrospray ionization (ESI) source working in a positive model and a negative model was used for accurate mass measurements. Liquid Chromatog- raphy analysis was performed using a Dionex UltiMate 3000 (Thermo Fisher Scientific, Bremen, Germany) equipped with a quaternary pump and a thermostated autosampler. Chromatographic separation was accomplished with an Agilent InfinityLab Poroshell 120, 4.6 mm × 150 mm, 2.7 µm column were used (Agilent Infinity Lab, Wilmington, DE, USA). The detailed operation was performed according to the previous method with slight modifications, and it is presented in the Supplementary Material [39].

2.6. Profiling of HC Constituents Compounds of HC were monitored using TraceFinder™ software v3.4 (Thermo Fisher Scientific, Waltham, MA, USA), loaded with the Orbitrap traditional Chinese medicine library (OTCML) database to process the UHPLC-MS data [40]. The OTCML database refers to the Chinese medicinal materials included in the Chinese pharmacopoeia (2015 edition), and it contains the mass spectrometry data and chromatographic retention time information of more than 1200 kinds of traditional Chinese medicine compounds and more than 7000 high-quality high-resolution secondary mass spectrometry data [41]. A threshold signal of 1.0 × 106 and an accurate mass measurement error lower than 5 ppm was established in the TraceFinder™ software to consider a positive match in the analyzed HC samples. In addition, compound confirmation was only granted if all the mentioned confirmation criteria (M/Z, isotopic pattern, and fragment ion) were accomplished after raw data processing with the TraceFinder™ screening software.

2.7. Collection of Candidate Targets For the targets of HC identiﬁed compounds, the databases of Traditional Chinese Medicine System Pharmacology (TCMSP) [42], Swiss Target Prediction [43], SymMap [44], and PubChem [45] were adopted to screen the candidate targets. For targets of sleep- related indication, the candidate targets were collected from the database of GeneCards (version 4.14) [46], DrugBank (version 5.1.6, released 22 April 2020) [47], and DisGeNET (version 7.0) [48–50]. The score of the gene symbol for the GeneCards database was set at higher than 5.0, and the score of the gene symbol for the DisGeNET database was set at higher than 0.1. For the DrugBank database, keywords for sleep-related indications were “insomnia”, “sleep disorders and disturbance”, “rapid eye movement sleep disorder”, “non-24-h sleep-wake disorder”, “disturbed sleep nightmares”, “sleep initiation disorders”, “shift-work related sleep disturb”, and “difﬁculty sleeping”. Foods 2021, 10, 883 4 of 15

2.8. Functional Enrichment Analysis and Network Construction Functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms in biological process (BP), molecular function (MF), and cellular components (CC) from overlapped targets were performed by using STRING bioinformatics resources and the KEGG database [51]. To uncover the complex interaction among targets, compounds, and sleep-related pathway, a C-T-P network was constructed by using Cytoscape (version 3.6) [52]. In addition, the protein-protein interaction (PPI) network of D. melanogaster was constructed by intersection targets, and three topological indicators (Degree, Betweenness centralityand Closeness centrality) of the network were further analyzed by the Network Analyzer [53,54].

2.9. mRNA Expression The D. melanogaster were fed with the corresponding diets (normal, caffeine, caffeine + 2.5% HC) in constant darkness for 3 days (n = 50). Total RNA was extracted from the heads of D. melanogaster using Trizol (GBCBIO Technologies, Guangzhou, China), according to the manufacture’s protocol. A reverse transcription reagent kit (TaKaRa, Dalian, China) was then used to obtain cDNA, and the mRNA expression levels were determined by RT-PCR using TB Green Premix Ex TaqII (TaKaRa, Dalian, China). The measured genes included Akt1, Sod, Cat, and Ple, and the Rp49 was used for the endogenous control [55,56]. The primer sequences have been displayed in Table S1. The relative expression levels were calculated by the ∆∆Ct method.

2.10. Statistical Analysis SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis of the data. The Kolmogorov–Smirnov test was used to test the normality of data. If the data then fit a normal distribution, one-way analysis of variance was used to analyze statistically significant differences between groups. Otherwise, non-parametric tests (the Kruskal-Wallis test) were used. The data of locomotor activity are presented as mean ± standard error of the mean (SEM) for each group. When the variances are assumed to be consistent, the least significant difference (LSD) was used to analyze the differences between groups. When the variances were not assumed to be consistent, Tamhane’s T2 was used to analyze the differences between groups. It was considered statistically significant when p < 0.05. The statistical procedures of mRNA expression were consistent with the statistical procedures of measurement of locomotor activity (the Shapiro–Wilk normality test was used to test the normality of data). The data of mRNA expression are presented as mean ± standard deviation (SD) for each group.

3. Results and Discussion 3.1. Effect of HC on Locomotor Activity of D. melanogaster Fruit flies, like humans, have periodic circadian rhythms and are a well-studied model organism for studying sleep. Meanwhile, caffeine has the same effect on reducing sleep duration in flies as it does in humans [19]. Therefore, in this study, the caffeine-induced insomnia model was used to investigate the effect of HC on locomotor activity in D. melanogaster by using the infrared beam-based Drosophila activity monitor (DAM) system. Figure1 provides an overview of the locomotor activities of each group of D. melanogaster over a 3-day period. To investigate the effects of HC on locomotor activity of D. melanogaster, the number of nighttime activity and the number of daytime activity was further analyzed (Figure2). The results revealed that a decreased tendency of nighttime activity was found in a different dose of HC. Among them, HC-H significantly decreased in the nighttime activity of D. melanogaster (compared to the caffeine group, p < 0.05) on day 1. On day 2, all HC groups significantly decreased in the nighttime activity of D. melanogaster (compared to the caffeine group, p < 0.05). A similar tendency of nighttime activity was found on day 3. For the amount of daytime activity, compared to the normal group, almost all groups (except the HC-L group on day 1) decreased in the amount of daytime activities from day 1 Foods 2021, 10, x FOR PEER REVIEW 5 of 17

in the nighttime activity of D. melanogaster (compared to the caffeine group, p < 0.05) on day 1. On day 2, all HC groups significantly decreased in the nighttime activity of D. mel- Foods 2021, 10, 883 anogaster (compared to the caffeine group, p < 0.05). A similar tendency of nighttime5 of ac- 15 tivity was found on day 3. For the amount of daytime activity, compared to the normal group, almost all groups (except the HC-L group on day 1) decreased in the amount of daytime activities from day 1 to day 3 (p < 0.05). However, no significant difference was tofound day 3in ( pall< HC 0.05). groups However, in comparison no signiﬁcant with difference the caffeine was group. found These in all HCresults groups suggest in com- that parisonHC could with effectively the caffeine decrease group. the These nighttime results activity suggest of that D. melanog HC couldaster effectively in a dose decrease-depend- theent nighttimemanner, while activity the of daytimeD. melanogaster activityin of a D. dose-dependent melanogaster was manner, not significantly while the daytime altered activitywhen caffeine of D. melanogaster was administered.was not signiﬁcantly altered when caffeine was administered.

FigureFigure 1.1. The effect of of HC HC on on the the overview overview of of locomotor locomotor activities activities in inD.D. melanogaster melanogaster duringduring the the3- 3-dayday intervent interventionion period. period. Black Black represents represents night night activity, activity, andand gray gray represents represents daytime daytime activity. activity. Normal:Normal: agar-sucroseagar-sucrose food;food; Caffeine:Caffeine: agar-sucroseagar-sucrose foodfood containingcontaining 0.1%0.1% caffeine;caffeine; CaffeineCaffeine ++ HC-L:HC-L: agar-sucrose food containing 0.1% caffeine + 0.625% HC extract; Caffeine + HC-M: agar-sucrose agar-sucrose food containing 0.1% caffeine + 0.625% HC extract; Caffeine + HC-M: agar-sucrose food food containing 0.1% caffeine + 1.250% HC extract; Caffeine + HC-H: agar-sucrose food containing containing 0.1% caffeine + 1.250% HC extract; Caffeine + HC-H: agar-sucrose food containing 0.1% 0.1% caffeine + 2.500% HC extract. caffeine + 2.500% HC extract.

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FigureFigure 2. 2. TheThe effect effect of of HC HC on on locomotor locomotor activities in D. melanogaster.melanogaster. Caffeine:Caffeine: 1.01.0mg/mL mg/mL ofof media;media;HC-L: HC- 0.625%L: 0.625% HC HC extract; extract; HC-M: HC-M: 1.250% 1.250% HC HC extract; extract; HC-H: HC-H: 2.500%2.500% HCHC extract. * * pp << 0.05 0.05 and and **** pp << 0.01 0.01 thanthan in in the the normal normal group; group; # # pp << 0.05 0.05 and and ## ## pp << 0.01 0.01 than than in in the the caffeine caffeine group. group. Values Values are are meanmean ±± standardstandard error error of of the the mean mean (SEM (SEM).).

3.2.3.2. Effect Effect of of HC HC on on Sl Sleepeep Behaviour Behaviour of of D. D. Melanogaster melanogaster ForFor the the total sleep sleep time, time, compared compared with with the the normal normal group, group, caffeine caffeine effectively effectively reduced re- ducedthe total the sleep total timesleep of timeD. melanogaster of D. melanogaster, indicating, indicating the insomnia the insomnia model was model successfully was success- built fully(Figure built3). In(Figure addition, 3). In we addition, found that we HC found elevated that HC the totalelevated sleep the time total of D.sleep melanogaster time of D.in melanogastera dose-dependent in a dose manner-dependent over amanner 3-day period.over a 3 In-day particular, period. In all particular, HC groups all signiﬁcantly HC groups significantlyimprove the improve total sleep the timetotal ofsleepD. melanogastertime of D. melanogasterin comparison in comparison with the caffeine with the group caffeine(day group 1–day (day 3, p < 1 0.05),–day 3, indicating p < 0.05), that indic HCating could that prolong HC could the prolong sleep time the of sleepD. melanogaster time of D. melanogasterinduced by caffeine.induced by caffeine. TheThe number number of of sleep sleep bouts bouts and and sleep sleep duration duration of D. of melanogasterD. melanogaster duringduring the night the night can providecan provide some some indication indication of sleep of sleep quality quality [13]. [Compared13]. Compared with withthe caffeine the caffeine group, group, all HC all groupHC groupss reduced reduced the number the number of sleep of sleepbouts, bouts, while whileonly that only of that the ofHC the-M HC-M group groupwas obvi- was ouslyobviously decreased decreased on day on day1 (Figure 1 (Figure 3). 3Meanwhile,). Meanwhile, we we found found that that all all HC HC group groupss elevated thethe sleep sleep duration duration more more than than that that of of the the caffeine caffeine group group (Figur (Figuree 33).). Of them, there was a statisticalstatistical difference difference between between the the HC HC-M-M group group and and the the caffeine caffeine group group (p (

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FigureFigure 3. 3. TheThe effect effect of of HC HC on on the the sleep sleep-wake-wake behavior behavior of of D.D. melanogaster melanogaster. Caffeine:. Caffeine: 1.0 1.0 mg/mL mg/mL of of media;media; HC HC-L:-L: 0.625% 0.625% HC extract; HC-M:HC-M: 1.250%1.250% HCHC extract;extract; HC-H:HC-H: 2.500% 2.500% HC HC extract. extract. * *p p< < 0.05 0.05 and and** p **< 0.01p < 0.01 than than in the in normalthe normal group; group; # p <# 0.05p < 0.05 and and ## p ##< 0.01p < 0.01 than than in the in the caffeine caffeine group. group. Values Val- are ues are mean ± SEM. mean ± SEM.

3.3.3.3. UHPLC UHPLC-ESI-Orbitrap-MS-ESI-Orbitrap-MS Analysis Analysis of of HC HC and and Compound Compound Identification Identification ToTo i identifydentify the compoundscompounds ofof HC, HC, the the optimized optimized LC-MS LC-MS conditions conditions were were adopted adopted for forthe the characterization characterization of HC of chemical HC chemical compounds. compounds. Furthermore, Furthermore both positive, both ESI positive modes ESIand modesnegative and ESI negative modes were ESI modes used to were identify used compounds to identify of compounds HC as comprehensively of HC as comprehen- as possible sively(Figures as possible S1 and S2). (Figure By importings S1 and S2). the By data importing into Tracefinder the data 3.4,into loaded Tracefinder with the3.4, OTCML loaded withdatabase, the OTCML the components database, of the HC components were identified. of HC Those were components identified. owned Those identicalcomponentsm/z , ownedfragment identical ions, and m/z isotopes, fragment to ions, the reference and isotope compounds.s to the reference As a result, compounds. 57 compounds As a result, of HC 57were compounds identified of from HC the were OTCML identified database, from mainlythe OTCML including database, flavonoids mainly (28), including phenolic flavo- acids noids(10), nucleotides(28), phenolic (8), aminoacids (10), acids nucleotides (5), and coumarins (8), amino (3). acid Thes specific (5), and matching coumarins information (3). The specificis summarized matching in Tableinformation1. In previous is summarized studies, Sun in Table et al. preliminary1. In previous identified studies, 27 Sun phenolic et al. preliminarycompounds iofdentified flowers of27 daylily phenolic (Hemerocallis compounds fulva of (L.)), flowers indicating of daylily that (Hemerocallis rutin, kaempferol- fulva (L.)),3-O-rutinoside, indicating that 5-O-caffeoylquinic rutin, kaempferol acid,-3-O and-rutinoside, 5-O-p-coumaroylquinic 5-O-caffeoylquinic acid acid were, and its major 5-O- pphenolic-coumaroylquinic compounds acid [39 were]. The its present major phenolic study performed compounds a comprehensive [39]. The present phytochemical study per- formedscan and a identified comprehensive more types phytochemical of plant compounds, scan and identifiednot limited more to polyphenols, types of plant which com- laid pounds,a firm basis not forlimited the subsequent to polyphenols, network which pharmacology laid a firm basis analysis. for the subsequent network pharmacology analysis.

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Table 1. Identiﬁcation of the chemical components of Hemerocallis citrina Baroni by UHPLC-ESI-Orbitrap-MS.

Molecular Measured Mass Compound Name RT (min) ESI-MS Accuracy (ppm) MS/MS (m/z) Category Formula (m/z) + 5-Hydroxymethylfurfural 1.61 C6H6O3 127.04 [M + H] −2.21 109.03, 81.03 Others + 2-Pyrrolidinecarboxylic acid 1.64 C5H9NO2 116.07 [M + H] −2.43 70.07 Amino acids + Cytosine 1.64 C4H5N3O 112.05 [M + H] −2.48 95.02, 69.04 Nucleotides − Uridine 2.25 C9H12N2O6 243.06 [M − H] −2.15 200.06, 152.03 Nucleotides + Adenosine 2.25 C10H13N5O4 268.1 [M + H] −2.72 136.06 Nucleotides + Guanine 2.25 C5H5N5O 152.06 [M + H] −1.83 135.03, 110.03 Nucleotides + Guanosine 2.25 C10H13N5O5 284.1 [M + H] −3.16 152.06 Nucleotides + L-Tyrosine 2.25 C9H11NO3 182.08 [M + H] −1.72 136.08, 123.04 Amino acids + Cordycepin 2.25 C10H13N5O3 252.11 [M + H] −2.5 136.06, 99.04 Nucleotides + Nicotinic acid 2.25 C6H5NO2 124.04 [M + H] −1.91 96.04, 80.05 Vitamin - Isoguanosine 2.27 C10H13N5O5 282.08 [M − H] −0.90 150.04, 107.03 Nucleotides + L-Leucine 2.8 C6H13NO2 132.1 [M + H] −1.59 86.1, 69.07 Amino acids − S-(-)-Carbidopa 2.83 C10H14N2O4 225.09 [M − H] −2.67 164.07 Phenolic acids − Thymidine 3.25 C10H14N2O5 241.08 [M − H] −1.26 151.05, 125.03 Nucleotides + L-Phenylalanine 3.95 C9H11NO2 166.09 [M + H] −1.21 149.06, 120.08 Amino acids + 7-Hydroxycoumarin 5.38 C9H6O3 163.04 [M + H] −1.21 107.05 Coumarins + Neochlorogenic acid 5.38 C16H18O9 355.1 [M + H] −2.07 163.04 Phenolic acids + 5-Acetylsalicylic acid 5.4 C9H8O4 181.05 [M + H] −1.57 107.05 Phenolic acids + Caffeic acid 5.4 C9H8O4 181.05 [M + H] −1.57 135.04, 117.03 Phenolic acids − 1-Caffeoylquinic acid 5.47 C16H18O9 353.09 [M − H] −0.55 191.06, 179.03 Phenolic acids − Chlorogenic acid 5.47 C16H18O9 353.09 [M − H] −0.55 191.06, 161.02 Phenolic acids − Cryptochlorogenic acid 5.47 C16H18O9 353.09 [M − H] −0.55 173.04, 135.04 Phenolic acids − Danshensu 6.24 C9H10O5 197.04 [M − H] −3.96 179.03, 135.04 Phenolic acids + L-Tryptophan 6.52 C11H12N2O2 205.1 [M + H] −1.33 146.06, 118.06 Amino acids + 4-Methylumbelliferone 8.68 C10H8O3 177.05 [M + H] −0.65 149.06 Coumarins + 7-Methoxycoumarin 8.68 C10H8O3 177.05 [M + H] −0.65 149.06, 121.06 Coumarins − Androsin 10.39 C15H20O8 327.11 [M − H] 0.50 165.05 Others + Grosvenorine 14.17 C33H40O19 741.22 [M + H] −1.60 287.05 Flavonoids − Typhaneoside 14.53 C34H42O20 769.22 [M − H] 0.95 314.04, 151.00 Flavonoids − Rutin 14.63 C27H30O16 609.15 [M − H] 0.31 300.03 Flavonoids − Taxifolin 14.81 C15H12O7 303.05 [M − H] 0.29 241.05 Flavonoids − Hyperoside 15.27 C21H20O12 463.09 [M − H] 0.72 300.03, 271.02 Flavonoids − Isoquercitrin 15.27 C21H20O12 463.09 [M − H] 0.72 300.03, 271.02 Flavonoids − Myricitrin 15.27 C21H20O12 463.09 [M − H] 0.72 271.02 Flavonoids + 5-Hydroxy-1-tetralone 16.72 C10H10O2 163.08 [M + H] −1.76 135.08, 107.05 Flavonoids + Oroxin B 16.76 C27H30O15 595.16 [M + H] −1.50 145.05 Flavonoids + Kaempferol 16.76 C15H10O6 287.05 [M + H] −2.40 153.02 Flavonoids + Luteolin 16.76 C15H10O6 287.05 [M + H] −2.40 153.02 Flavonoids Kaempferol 16.77 C H O 593.15 [M − H]− −0.42 327.05, 284.03 Flavonoids 3-glucorhamnoside 27 30 15 − Kaempferol-3-O-rutinoside 16.77 C27H30O15 593.15 [M − H] −0.42 285.04, 255.03 Flavonoids − Lonicerin 16.77 C27H30O15 593.15 [M − H] −0.42 285.04 Flavonoids Isorhamnetin-3-O- 17.32 C H O 623.16 [M − H]− 1.25 314.04, 271.03 Flavonoids nehesperidine 28 32 16 − Narcissoside 17.32 C28H32O16 623.16 [M − H] 1.25 315.05, 271.03 Flavonoids − Astragalin 17.45 C21H20O11 447.09 [M − H] 0.30 284.03, 227.03 Flavonoids − Cynaroside 17.45 C21H20O11 447.09 [M − H] 0.30 285.04, 151.00 Flavonoids − Homoorientin 17.45 C21H20O11 447.09 [M − H] 0.30 327.05 Flavonoids Kaempferol-7-O-β-D- 17.45 C H O 447.09 [M − H]− 0.30 285.04, 151.00 Flavonoids glucopyranoside 21 20 11 − Orientin 17.45 C21H20O11 447.09 [M − H] 0.30 327.05 Flavonoids − Quercetin 7-rhamnoside 17.45 C21H20O11 447.09 [M − H] 0.30 301.04 Flavonoids − Quercitrin 17.45 C21H20O11 447.09 [M − H] 0.30 300.03 Flavonoids − Eriodictyol 18.28 C15H12O6 287.06 [M − H] 0.57 151 Flavonoids + Dehydrodiisoeugenol 19.6 C20H22O4 327.16 [M + H] −2.52 203.11 Phenolic acids + Nepodin 20.49 C13H12O3 217.09 [M + H] −0.55 184.05, 171.08 Phenolic acids − Morin 21.21 C15H10O7 301.04 [M − H] −0.03 151, 107.01 Flavonoids − Quercetin 21.21 C15H10O7 301.04 [M − H] −0.03 179.00, 151.00 Flavonoids − Demethylwedelolactone 21.21 C15H8O7 299.02 [M − H] 0.25 271.02 Flavonoids − Myricetin 21.21 C15H10O8 317.03 [M − H] −0.44 179.00, 151.00 Flavonoids RT: retention time.

Nepodin 20.49 C13H12O3 217.09 [M + H]+ −0.55 184.05, 171.08 Phenolic acids Morin 21.21 C15H10O7 301.04 [M − H]− −0.03 151, 107.01 Flavonoids Quercetin 21.21 C15H10O7 301.04 [M − H]− −0.03 179.00, 151.00 Flavonoids Demethylwedelolactone 21.21 C15H8O7 299.02 [M − H]− 0.25 271.02 Flavonoids Myricetin 21.21 C15H10O8 317.03 [M − H]− −0.44 179.00, 151.00 Flavonoids RT: retention time.

3.4. Screening of the Candidate Targets As a complex system with multiple components, HC may play a role in promoting health through multiple targets and multiple pathways. Network pharmacology is a new strategy based on system pharmacology and bioinformatics [57], and thus it was applied to screen the candidate sleep-improvement compounds of HC. For the collection of potential targets for HC, a total of 2075 candidate targets (Homo sapiens) from the 56 active components of HC were selected by applying the database for TCMSP, Swiss Target Pre- Foods 2021, 10, 883 diction, SymMap, and PubChem, respectively (Table S2). For the collection of therapeutic 9 of 15 targets of sleep-related indications, 1132 targets associated with sleep-related indications were screened from the database for DrugBank, DisGeNET, and Genecards, respectively (Table S3).respectively Eventually,(Table a Venn S3) diagram. Eventually, was constructed a Venn diagram to visualize was constructed the intersection to visualize be- the inter- tween compoundsection betweentargets and compound sleep-related targets targets. and sleep-related As a result, targets. 323 overlapp As a result,ing targets 323 overlapping were screenedtargets (Figure were screened4, Table S4 (Figure and Table4, Tables S6), S4 indicating and S6), indicating323 candidate 323 candidatetargets of targetsHC of HC compoundscompounds were hit from were 1132 hit fromknown 1132 therapeutic known therapeutic targets of sleep targets-related of sleep-related indications. indications.

Figure 4. (a) The intersection targets of HC and sleep; (b) the protein-protein interaction (PPI) network of D. melanogaster constructed by intersection targets. The size of nodes represents the value of degree.

3.5. Functional Enrichment of GO Terms and KEGG Pathway To unveil the sleep-improvement mechanisms of HC, the top 10 GO terms for CC, BP, and MF were picked out based on the p-value (Figure5). For CC, the extracellular space, extracellular region part, extracellular region, neuron part, neuron projection, vesicle, cell body, axon, cytoplasmic vesicle, and somatodendritic compartment were signiﬁcantly enriched (p < 0.05). Furthermore, the results of GO terms showed that overlapping targets were enriched in the BP, including response to chemicals, regulation of biological quality, response to organic substances, response to oxygen-containing compounds, cellular response to chemical stimulus, response to stimulus, response to external stimulus, regulation of signaling, regulation of cell communication, and regulation of multicellular organismal processes (p < 0.05). For MF, signaling receptor binding, protein binding, binding, receptor ligand activity, receptor regulator activity, molecular function regulator, hormone activity, identical protein binding, cofactor binding, and enzyme binding were signiﬁcantly enriched (p < 0.05). Foods 2021, 10, 883 10 of 15 Foods 2021, 10, x FOR PEER REVIEW 11 of 17

FigureFigure 5. 5. FunctionalFunctional enrichment enrichment analysis analysis for for GO GO terms terms and and KEGG KEGG pathways pathways (Homo (Homo sapiens) sapiens):: (a) (a) CellularCellular Component Component,, ( (bb)) Biological Biological Pro Process,cess, ( (cc)) Molecular Molecular Function Function,, ( (dd)) KEGG KEGG pathways. pathways.

3.6. ConstructionTo gain further of the insight C-T-P Network into the relationship between sleep and HC, KEGG pathway enrichmentTo uncover analysis the interactions was carried among out. As identified a result, 10 compounds, remarkable overlapp pathwaysing related targets, to sleepand signalingwere selected, pathways, which a mainlyC-T-P network involved was the neuroactiveconstructed ligand-receptor(Figure S3). The interaction,results showed sero- thatonergict the C synapse,-T-P network relaxin consists signaling of 387 pathway, nodes and neurotrophin 1747 edges, signaling indicating pathway, HC possesses tryptophan the characteristicsmetabolism, dopaminergic of multi-compounds, synapse, cholinergic multi-targets, synapse, and circadian multi-pathways entrainment, for sleep circadian-improvement.rhythm, and In GABAergicparticular, three synapse important (p < 0.05). topological Among properties them, neuroactive of “Degree”, ligand-receptor “Between- neinteractionss centrality” was, theand most “Closeness important centrality” topological were pathway, further andanalyzed is closely to uncover associated the with im- portantneurological compounds function and [58 ].targets Furthermore, of HC in the sleep neurotransmitters-improvement for(Table serotonin,s 2 and GABA, S5). For acetyl- the target,choline, the and mean dopamine degree value play keyof all functions candidate in targets regulating was sleep5.41, and and the wakefulness network analysis [59–61]. showedThese results that Tnf further (degre reveale = 46), that Akt1 the (degre sleep-improvemente = 39), ins (degre effectse = 3 of8), HCil6 (degre involvee = multiple 37), cat (degretargetse and= 36), multiple alb (degre pathways.e = 35), sod1 (degree = 35), ache (degree = 33), ptgs2 (degree = 33), and3.6. tp Construction53 (degree of= 3 the3) C-T-Pwere the Network top 10 targets in the C-T-P network, indicating these targets of HC may be the key targets in sleep-improvement. For the compound, the mean To uncover the interactions among identiﬁed compounds, overlapping targets, and degree value of all identified compounds was 29.70, and the quercetin (degree = 127), lu- signaling pathways, a C-T-P network was constructed (Figure S3). The results showed that teolin (degree = 70), kaempferol (degree = 59), caffeic acid (degree = 59), nicotinic acid the C-T-P network consists of 387 nodes and 1747 edges, indicating HC possesses the char- (degree = 56), myricetin (degree = 55), chlorogenic acid (degree = 53), s-(-)-carbidopa (de- acteristics of multi-compounds, multi-targets, and multi-pathways for sleep-improvement. gree = 50), rutin (degree = 47), and hyperoside (degree = 45) were the top 10 identified

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In particular, three important topological properties of “Degree”, “Betweenness centrality”, and “Closeness centrality” were further analyzed to uncover the important compounds and targets of HC in sleep-improvement (Table2 and Table S5). For the target, the mean degree value of all candidate targets was 5.41, and the network analysis showed that Tnf (degree = 46), Akt1 (degree = 39), ins (degree = 38), il6 (degree = 37), cat (degree = 36), alb (degree = 35), sod1 (degree = 35), ache (degree = 33), ptgs2 (degree = 33), and tp53 (degree = 33) were the top 10 targets in the C-T-P network, indicating these targets of HC may be the key targets in sleep-improvement. For the compound, the mean degree value of all identified compounds was 29.70, and the quercetin (degree = 127), luteolin (degree = 70), kaempferol (degree = 59), caffeic acid (degree = 59), nicotinic acid (degree = 56), myricetin (degree = 55), chlorogenic acid (degree = 53), s-(-)-carbidopa (degree = 50), rutin (degree = 47), and hyperoside (degree = 45) were the top 10 identified compounds in terms of degree, and they may be the key compounds of HC in sleep- improvement because of their crucial positions in the C-T-P network. Previous studies have reported that quercetin and rutin can alter the sleep-wake cycle by activating GABA(A) receptors [29,62], and luteolin has hypnotic efficacy through ADORA1 and ADORA2A binding [63]. Both caffeic acid and chlorogenic acid significantly prolong sleep latency in rats [33], and nicotinic acid promotes sleep through prostaglandin synthesis [64]. Further- more, myricetin modulates the GABA(A) receptor activity [65], and hyperoside shows an inhibitory effect on the central nervous system by mediating the dopaminergic system [30]. All of these findings provide further evidence for our results that quercetin, luteolin, kaempferol, caffeic acid, nicotinic acid, myricetin, chlorogenic acid, s-(-)-carbidopa, rutin, and hyperoside are the main active compounds of HC in sleep-improvement.

Table 2. The C-T-P network topology analysis for top 20 compounds.

Betweenness Compound Degree Closeness Centrality Centrality Quercetin 127 0.26 0.46 Luteolin 70 0.06 0.40 Kaempferol 59 0.04 0.39 Caffeic acid 59 0.06 0.39 Nicotinic acid 56 0.07 0.39 Myricetin 55 0.03 0.39 Chlorogenic acid 53 0.04 0.39 S-(-)-Carbidopa 50 0.09 0.38 Rutin 47 0.03 0.38 Hyperoside 45 0.03 0.38 Adenosine 45 0.06 0.38 Myricitrin 43 0.06 0.38 Danshensu 42 0.03 0.38 Isoquercitrin 40 0.01 0.38 Thymidine 39 0.03 0.38 Cordycepin 39 0.03 0.38 L-Tryptophan 36 0.04 0.37 7-Hydroxycoumarin 35 0.01 0.38 Kaempferol-3-O- 33 0.02 0.37 rutinoside Cynaroside 33 0.01 0.37

3.7. Effects of HC on mRNA Levels in D. melanogaster To further validate the core targets of the Drosophila-associated PPI networks affected by HC, the top 4-degree targets (Akt1, Sod, Cat, and Ple) were selected (Figure4b). In this study, the transcript levels for the Akt1 in the caffeine group were signiﬁcantly higher than for the normal group (Figure6). However, HC treatment signiﬁcantly reduced the level of Akt1. The previous study reported that Akt pathways are a major regulator of nutrient metabolism, cell growth, and senescence, and they impact the brain circadian Foods 2021, 10, 883 12 of 15

clock that drives behavioral rhythms in D. melanogaster [66] and the rhythmic expression of Akt1 in the skeletal muscle of mice [67]. These results suggest that HC can ameliorate the changes in Akt1 transcription levels induced by caffeine. In addition, the D. melanogaster exposed to caffeine had signiﬁcantly decreased transcript levels for Cat, Ple, and Sod (Figure6), while the administration of HC effectively reversed this trend ( p < 0.05). The Foods 2021, 10, x FOR PEER REVIEW previous studies have reported the effects of some ﬂavonoids on the temporal regulation13 of 17 of redox homeostasis [68,69]. Among them, hesperidin increased the levels of the antioxidant index (Sod and Cat) and regulated their amplitudes in the rotenone-induced oxidative stress model of Drosophila [69]. Furthermore, the synthesis of dopamine was regulated locomotorby expression behaviors of Ple and [70], arousal and dopamine states [71 has,72 several]. Therefore, roles HC in the positively modulation regulated of locomotor the ex- pressionbehaviors level and of arousal Sod, Cat states, and [ 71Ple,72 in]. the Therefore, head of HCD. melanogaster positively regulated to play a the role expression in sleep- improvement.level of Sod, Cat, and Ple in the head of D. melanogaster to play a role in sleep-improvement.

FigureFigure 6. 6. TheThe effect effect of of HC HC on on core core target target mRNA mRNA expression in the heads of D.D. melanogastermelanogaster.. Caffeine:Caf- feine:1.0 mg/mL 1.0 mg/mL of media; of media; HC-H: HC 2.500%-H: 2.500% HC extract.HC extract. * p < * 0.05 p < 0.05 and **andp <** 0.01 p < 0.01 than than in the in normalthe normal group; group;# p < 0.05 # p and< 0.05 ## andp < ## 0.01 p < than 0.01 in than the in caffeine the caffeine group. group. Values Values are mean are ±meanstandard ± standard deviation deviation (SD). (SD). 4. Conclusions 4. ConclusionsIn this work, the results suggested that HC extract effectively regulated the number of nighttimeIn this activitieswork, the and results total suggested sleep time that of D.HC melanogaster extract effectivelyin a dose-dependent regulated the number manner ofand nighttime positively activities regulated and the total sleep sleep bouts time and of sleepD. melanogaster durationof inD. a dose melanogaster-dependent. In addition,manner andthe p chemicalositively constituentsregulated the of sleep HC werebouts full-scale and sleep identified duration of by D. UHPLC-ESI-Orbitrap, melanogaster. In addition, and the57 compoundschemical constituents were identified. of HC Amongwere full them,-scale C-T-P identified network by UHPLC topology-ESI analysis-Orbitrap, revealed and 57that compounds quercetin, luteolin,were identified. kaempferol, Among caffeic them, acid, C- nicotinicT-P network acid, topology myricetin, analysis chlorogenic revealed acid, thats-(-)-carbidopa, quercetin, luteolin, and rutin kaempferol, were the key caffeic compounds acid, of nicotinic HC in sleep-improvement. acid, myricetin, chlorogenic Moreover, acid,the core s-(- targets)-carbidopa, (Akt1, and Sod, rutin Cat, andwere Ple) the of key the Drosophila-associatedcompounds of HC in PPIsleep networks-improvement. affected Moreover,by HC were the verified core targets by transcriptional (Akt1, Sod, Cat levels., and In Ple) a word, of the this Drosophila study revealed-associate thatd HC PPI plays net- worksa beneficial affected role by in HC sleep were improvement verified by throughtranscriptional multiple levels. compounds, In a word, multiple this study targets, re- and multiple pathways, which provides a new perspective on the active components and vealed that HC plays a beneficial role in sleep improvement through multiple com- molecular mechanism of HC. pounds, multiple targets, and multiple pathways, which provides a new perspective on the active components and molecular mechanism of HC. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/foods10040883/s1, Figure S1: Base peak chromatograms of compounds from HC by UPLC- Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Orbitrap-MS in ESI−, Figure S2: Base peak chromatograms of compounds from HC by UPLC- Base peak chromatograms of compounds from HC by UPLC-Orbitrap-MS in ESI−, Figure S2: Base Orbitrap-MS in ESI+, Figure S3: The Component-Target-Pathway (C-T-P) network (Homo sapiens). peak chromatograms of compounds from HC by UPLC-Orbitrap-MS in ESI+, Figure S3: The Com- Blue hexagons represent top 10 pathways, green diamonds represent components of HC, and red ponent-Target-Pathway (C-T-P) network (Homo sapiens). Blue hexagons represent top 10 pathways, greenrounds diamonds represent represent overlapped components targets, Table of HC S1:, RT-PCRand red primerrounds designrepresent sequence, overlapped Table targets, S2: Candidate Table S1:targets RT-PCR for HC,primer Table design S3: Therapeuticsequence, Table targets S2: Candidate for sleep relatedtargets indications,for HC, Table Table S3: Therapeutic S4: Intersection tar- getsbetween for sleep targets rela ofted HC indications, and targets Table of sleep, S4: Intersection Table S5: The between C-T-P targets network of topologyHC and target analysiss of forsleep, top Table20 targets, S5: The Table C-T S6:-P network The PPI networktopology of analysisD. melanogaster for top 20constructed targets, Table by intersection S6: The PPI targets. network of D. melanogasterAuthor Contributions: constructed Conceptualization,by intersection targets. Y.L. and Y.S.; methodology, R.H.; software, Y.C.; valida- Authortion, Y.L.; Contribution formal analysis,s: Conceptualization, Y.L.; investigation, Y.L. Z.W. and (ZiyiY.S.; methodology, Wang) and Z.W. R.H. (Zhuojun; software, Wu); Y.C. resources,; valida- tion, Y.L.; formal analysis, Y.L.; investigation, Z.W. (Ziyi Wang) and Z.W. (Zhuojun Wu); resources, Y.C.; data curation, J.D. and J.Z.; writing—original draft preparation, Y.L.; writing—review and editing, H.W.; visualization, Y.L.; supervision, M.L.; project administration, R.H.; funding acquisition, H.W. and Y.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Key Realm R&D Program of Guangdong Province (2019B020211002), the National Natural Science Foundation of China (31972157), the Science and Technology Planning Project of Guangzhou City (201804020077), the International Cooperation Pro- gram of SCAU (2019SCAUGH03), and Project Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017).

Foods 2021, 10, 883 13 of 15

Y.C.; data curation, J.D. and J.Z.; writing—original draft preparation, Y.L.; writing—review and editing, H.W.; visualization, Y.L.; supervision, M.L.; project administration, R.H.; funding acquisition, H.W. and Y.S. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Key Realm R&D Program of Guangdong Province (2019B020211002), the National Natural Science Foundation of China (31972157), the Science and Technology Planning Project of Guangzhou City (201804020077), the International Coop- eration Program of SCAU (2019SCAUGH03), and Project Supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or supplementary material. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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